Immunogenicity of poxvirus-based vaccines against Nipah virus

Nipah virus (NiV), an emerging zoonotic pathogen in Southeast Asia, is transmitted from Pteropus species of fruit bats to a wide range of species, including humans, pigs, horses, dogs, and cats. NiV has killed millions of animals and caused highly fatal human outbreaks since no vaccine is commercially available. This study characterized the immunogenicity and safety of poxvirus-based Nipah vaccines that can be used in humans and species responsible for NiV transmission. Mice were vaccinated with modified vaccinia Ankara (MVA) and raccoon pox (RCN) viral vectors expressing the NiV fusion (F) and glycoprotein (G) proteins subcutaneously (SC) and intranasally (IN). Importantly, both vaccines did not induce significant weight loss or clinical signs of disease while generating high circulating neutralizing antibodies and lung-specific IgG and IgA responses. The MVA vaccine saw high phenotypic expression of effector and tissue resident memory CD8ɑ+ T cells in lungs and splenocytes along with the expression of central memory CD8ɑ+ T cells in lungs. The RCN vaccine generated effector memory (SC) and tissue resident (IN) CD8ɑ+ T cells in splenocytes and tissue resident (IN) CD8ɑ+ T cells in lung cells. These findings support MVA-FG and RCN-FG viral vectors as promising vaccine candidates to protect humans, domestic animals, and wildlife from fatal disease outcomes and to reduce the global threat of NiV.

Expression cassette designs. The MVA gene cassette (Fig. 1A) contained the F and G protein sequences from the 2004 Bangladesh strain of NiV (GenBank: AY988601). The strong pHyb promoter was placed in-frame with the full-length F gene containing a natural secretory signal to drive its expression, and the modified early/ late PrH5m promoter (H5 gene from vaccinia virus) was used to control the expression of the G gene in frame with the tissue plasminogen activator (tPA) secretory signal 33 . The mCherry fluorescence marker (GenBank: ANF29837) and the xanthine guanine phosphoribosyl transferase (GPT) selection marker (Escherichia coli, GenBank: 75205729) were included to facilitate the selection of recombinant viruses.
For the RCN expression cassette (Fig. 1B), the F and G protein sequences from the 2018 NiV isolated during an outbreak in Kerala, India (GenBank: MH523642) were used. The F protein was fused with the human IgG secretory signal after removing the natural virus-derived secretory signal and expressed under the control of the PrMVA13.5L promoter (naturally occurring promoter in vaccinia virus; GenBank: MT227314). Similarly, the G protein was fused with the tPA secretory signal, and the hybrid PrS5E promoter was used to control its expression. The inclusion of the eGFP protein (GenBank: MN623123) permitted an easy distinction between wild-type (no fluorescence) and recombinant (green) viruses. Mouse studies. While hamsters are typically used as a model for NiV animal studies, mice were chosen due to ease of handling and as challenge studies were not performed, a species susceptible to Nipah virus was not needed. BALB/c and AJ mice (4-week-old mixed sex) were purchased from Jackson Laboratory (JAX, Sacramento, CA, USA) and housed in a specific-pathogen-free room at the BSL-II Animal Health and Biomedical Sciences building per UW-Madison husbandry protocols. Mice were anesthetized prior to vaccination and blood collection via isoflurane inhalation in a closed-chamber system. Mice were divided into six groups, with eight mice of mixed sex per group (Fig. 2B). After an acclimatization period of 7 days, three groups received the MVA-FG vaccine (BALB/c mice), while the remaining three groups received the RCN-FG vaccine (AJ mice). Groups received one dose via subcutaneous injection (SC) or intranasal (IN) for each vaccine or two doses via SC 28 days apart ( Fig. 2A). Viruses were diluted in 1 mM Tris-HCL, and mice were given a dose of 10 8 PFU for each immunization (50µL per mouse). At weeks 3, 5, and 7 after prime a submandibular blood puncture was performed using a 4 mm animal lancet. Samples were collected in Microvette Ⓡ 500 Z-Gel (Sarstedt, VWR, PA, Cat. No.: 103218-893), kept at RT for 15 min, and centrifuged at 10,000g at 20 °C for 5 min. Serum was allocated and stored at − 20 °C for ELISA and neutralization assays. After euthanizing the animals 7 weeks after prime, spleen and lung cells were collected to assess the level of F and G-specific cytokine-secreting cells via ELISpot. Daily observation for any changes in health status by UW-Madison veterinary car staff and weighing three times per week for 2 weeks were carried out after vaccination to monitor changes in the animal's body condition.
For flow cytometry studies, vaccines were prepared as described previously, and two doses of 10 8 PFU for each immunization were given four weeks apart (Fig. 3A). BALB/c mice were divided into three groups, with four mice per group receiving MVA-FG via SC and IN, and MVA-GFP via SC. AJ mice were similarly divided and received RCN-FG via SC and IN, and RCN-WT via SC (Fig. 3B). Serum was collected 7 weeks after prime to assess the humoral immune response of boosted IN groups via ELISA. At 8 weeks post-prime, T cell memory responses were measured in spleen and lung cells and a bronchoalveolar lavage was performed to evaluate the level of F and G specific IgG, IgA, and IgM antibodies in lung tissues.     37 . Wells were examined for cytopathic effects (CPE) after 6 days p.i. Reciprocal dilution titers were considered neutralizing at the lowest dilution in which both duplicates showed no CPE. Naive sera from BALB/c and AJ mice were tested as negative controls, and rabbit anti-Hendra soluble G serum was used as a positive control (neutralizing up to a reciprocal dilution of 640) 38 . Anti-Hendra G serum was used as a positive control due to its ability to elicit neutralizing antibody responses against both Nipah and Hendra viruses 39 . Viruses were back-tittered in duplicate for virus stocks to confirm intended inoculum amount. Following humane euthanasia, mice were pinned, and the neck was disinfected with 70% ethanol. An incision was made at the neck, and skin was pulled back to expose the trachea. A 20 G catheter needle was then inserted above the cartilage rings of the trachea to a depth of 0.5 cm. A 1 mL syringe loaded with 1% FBS in Roswell Park Memorial Institute medium (RPMI) was injected into the lungs, and the aspirated fluid was collected in a 15 mL conical tube. After repeating this process three times, the BALF fluid was centrifuged at 800g for 3 min at 4 °C. The supernatant was collected and stored at − 20 °C until analysis via ELISA.

Spleen
ELISpot IFN-γ, IL-2, and TNF-α analysis. Spleen and lung cells were added in duplicate with 300,000 cells/well to high-protein-binding PVDF 96-well plates primed with murine IFN-γ/IL-2 capture solution or TNF-ɑ capture solution according to the manufacturer's protocol. Purified NiV-F (Native Antigen) and NiV-G (Native Antigen) antigens were added at 0.2 µg/well in duplicate and incubated at 37 °C for 48 h. Plates were then washed, incubated with corresponding cytokine detection solutions, and developed according to the manufacturer's protocol. Spots were scanned with ImmunoSpot software (Version 7.0.15.2) and counted using the open-source software Viridot 40 .
T cell memory assessed by flow cytometry. Splenocytes and lung cells were counted and seeded at around 2 × 10 6 cells/well into 96-well round-bottom plates. Plates were centrifuged at 800g for 3 min at RT, and the supernatant was discarded. For each tissue type and animal, the cells were stimulated with 200 µL/well of an F protein-peptide pool (13-mers with 2 amino acid overlap, ThermoFisher), a G protein-peptide pool (13-mers with 2 amino acid overlap, ThermoFisher), a cell stimulation cocktail (TONBO, Cat. No.: TNB-4975) as a positive control, or dimethyl sulfoxide (DMSO)/RPMI-complete medium (Lonza, Cat. No.:BE12-115F) as a negative control. Plates were incubated for 5 h at 37 °C, 5% CO 2 . After incubation, plates were centrifuged at 800g for 3 min at 4 °C and washed with 1X PBS where supernatant was discarded. A 1:1000 live/dead cell stain (Aqua-Dye, ThermoFisher) diluted in 1X PBS was mixed with cells and incubated for 30 min at 4 °C in the dark. Cells were then washed with FACS buffer (1X PBS, 0.5% BSA, 0.1% sodium azide) and centrifuged at 800g for 3 min at 4 °C, where the supernatant was discarded. Surface staining antibodies (Supplementary Table 1

Flow cytometry gating strategy
Compensation was done using UltraComp eBeads Compensation Beads (ThermoFisher, Cat. No.: A10344) and an ArC Amine Reactive Compensation Bead Kit (Thermo Fisher Scientific, Cat. No.: 01-2222-42). Data were analyzed using FlowJo software (BD Biosciences, TreeStar, Ashland, OR, USA) and an automated flowClean FlowJo plugin 41 . Gating strategies (Supplementary Fig. 1) excluded dead cells using a cell viability stain (Aqua Dye), doublet exclusion via forward scatter height (FCS-H) and forward scatter area (FCS-A) dot plot and selected for proliferating CD4 + and CD8α + T cells using a forward (FSC) and side scatter (SSC) dot plot. Live, CD4 + and CD8α + lymphocytes were then gated positive at the FITC axis and the BUV395 axis, respectively, in a FITC-BUV395 dot plot. Similar approaches were used to identify populations of CD4 + and CD8α + T cells expressing cell surface markers. Samples were excluded from analysis for tissue samples yielding non-viable cell isolates.

Statistical Analysis
Statistical analysis was performed between vaccine delivery groups for antibody titers using a one-way Kruskal Wallis Analysis of variance (ANOVA) and assessed using a Dunn's multiple comparison test upon discovery of significant differences. Flow cytometry analysis was conducted using a one-way ANOVA and a Tukey's multiple comparison test after the discovery of significant differences. All data were analyzed with GraphPad Prism version 9.1.1 software for Mac, GraphPad Software, San Diego, California USA, www. graph pad. com. P-values < 0.05 (alpha) were considered statistically significant.

Production and in vitro characterization of viral vector vaccines.
The presence of the F and G genes, including the mCherry and eGFP genes, with their corresponding promoters and regulatory sequences, was confirmed via PCR and cell culture for both MVA-FG and RCN-FG virus constructs. DNA sequencing indicated no genetic alterations in the final virus stocks (data not shown).
We assessed the expression and cellular localization of the F and G proteins by immunofluorescence microscopy of MVA-FG and RCN-FG-infected cells (Fig. 4). F protein expression was identified in permeabilized (  While the RCN-FG vaccine displayed the formation of syncytia and both F and G protein localization on the cell membrane was confirmed via immunofluorescence, the same could not be said for the MVA-FG vaccine. It is hypothesized based on the lack G protein localization via immunofluorescence, lack of syncytia formation in infected cells, and the detection of the G protein in the western blot, that the G protein is being secreted into the supernatant and is not membrane-anchored for the MVA-FG vaccine.

Post-immunization safety monitoring of mice vaccinated with MVA-FG and RCN-FG
Mice were vaccinated with one or two doses via SC and one dose via IN immunization with 10 8 PFU of either MVA-FG or RCN-FG vaccine candidates. Animals were weighed three times per week for a period of two weeks and monitored daily for any changes in health status by UW-Madison animal care staff. Mouse body weights postvaccination (p.v.) were compared to the baseline weight taken immediately prior to vaccination. Interestingly, the only group that showed a decrease in weight were mice immunized IN with MVA-FG (Fig. 5). MVA-FG IN mice lost less than 5% of body weight compared to their baseline weight following vaccination until day 5 p.v. After 5 days p.v, MVA-FG IN mice began increasing in weight until day 10 where they surpassed their initial baseline weight. RCN-FG vaccine groups and MVA-FG groups vaccinated SC had a steady increase in weight p.v. throughout the two-week period. The limited weight loss p.v. and no adverse health events reported by veterinary care staff corroborate previous viral vector safety data and provide initial safety data for these vaccine candidates to undergo further studies for rigorous safety testing.

NiV-F and NiV-G specific antibody levels induced in mice
Mice were vaccinated with either MVA-FG or RCN-FG viral vectors using one or two doses via SC injection or one dose via IN injection (Fig. 2). The reciprocal endpoint IgG antibody titers were measured using ELISA from serum samples collected on weeks 3, 5, and 7 (Fig. 6). Antibody titers were expressed as the logarithm base 10 geometric mean titer (GMT) per group. In terms of anti-F antibody titers (Fig. 6A), there were no statistical differences between all MVA-vaccinated groups. All three groups generally induced low levels at week 3, which increased when measured at weeks 5 and 7. Specifically, the SC boost group showed higher F titer levels at weeks 5 and 7 than the SC prime and IN groups.
In contrast, RCN-vaccinated groups displayed differences in F antibody titers (Fig. 6A). Overall, the IN group consistently induced higher levels at all three-time points than the other two groups. On the other hand, both SC groups showed an increase in titer levels from week 3 to week 7, but lower than IN groups. Statistical differences at weeks 3 and 5 indicated that the IN group induced higher levels than SC prime and SC boost at week 3. However, there were no differences between IN and SC prime groups at week 7 and between IN and SC boost at weeks 5 and 7. Notably, F titers from animals immunized with RCN-FG IN were higher than levels observed in all three MVA-vaccinated groups (Fig. 6A).
Overall, the G-specific antibody titers induced in mice vaccinated with MVA-FG or RCN-FG (Fig. 6B) were higher than antibody titers seen against the F protein. BALB/c mice immunized with the MVA-FG vaccine www.nature.com/scientificreports/ showed statistically comparable G antibody titers among all MVA-vaccinated groups but with a higher mean antibody titer seen in groups vaccinated SC versus IN. High G antibody titers were achieved at all three weeks tested for the IN-treatment group for RCN-FG vaccinated AJ mice. Interestingly, titer levels induced in the SC boost group peaked at week 5 and were similar to the IN group but showed a reduction at week 7 comparable to levels obtained at week 3. In contrast, the SC prime group peaked at week 3 but gradually induced lower levels at weeks 5 and 7. F specific antibody titers for MVA-FG and RCN-FG IN boosted mice (Fig. 6C) saw an increase at 7 weeks post-prime compared to mice receiving a single dose. Antibody titers against the G protein showed comparable levels between mice receiving one or two doses of either the MVA-FG or the RCN-FG vaccines.
In the flow cytometry T cell memory study (Fig. 3), serum was collected from BALB/c and AJ mice vaccinated with two doses of either MVA-FG or RCN-FG IN four weeks apart and the levels of circulating antibodies 7 weeks after prime for IN groups were measured. F antibody titers in MVA-FG and RCN-FG vaccinated mice showed an increase in the mean antibody titer following an IN boost, while G mean antibody titers remained comparable to levels after a single dose for both MVA-FG and RCN-FG-vaccinated mice.
Both vaccines produced high levels of circulating antibodies against both the F and the G glycoproteins and were assessed based on vaccine administration route. Mice vaccinated with MVA-FG did not have antibody titer differences when using different administration routes suggesting route of administration could be variable. Unlike MVA-FG vaccinated mice, RCN-FG vaccinated mice did show differences in antibody production levels based on vaccine administration route, suggesting the IN vaccine delivery route would outperform the SC route in generating circulating antibodies.

Neutralizing antibodies induced in mice
Mouse serum collected on weeks 3, 5, and 7 was evaluated for the presence of nAbs against the NiV Bangladesh strain (Fig. 7). Serum was tested in duplicate for 100% neutralization via CPE visualization 6 days p.i. Reciprocal dilution titers were considered neutralizing at the lowest dilution in which both duplicates showed no CPE. Although none of the vaccine candidates induced nAbs at week 3 after the first immunization, in the MVA-FG vaccinated animals, the SC boost group induced the highest mean titer of nAbs, which increased from week 5 Overall, both vaccines were able to induce neutralizing antibodies. For mice vaccinated with MVA-FG, the level of neutralizing antibodies produced was dependent on vaccine administration route with the SC groups having significantly higher neutralizing antibodies the IN group. Furthermore, mice vaccinated SC also showed an increase in the level of neutralizing antibodies generated with two doses versus a single dose. Therefore, while circulating antibodies were unaffected by vaccine administration route, the ability of the antibodies to neutralize the virus was affected. Mice vaccinated with RCN-FG had differences like the overall level of circulating antibodies seen in serum. The RCN-FG IN group had significantly high levels of neutralizing antibodies generated quickly compared to SC administration, affirming the IN group as the better vaccine administration route. Serum samples were collected on weeks 3, 5, and 7 following the initial vaccination. (C) Serum antibody titers (GMT ± GSD) in BALB/c and AJ mice that were vaccinated four weeks apart with two doses of either MVA-FG or RCN-FG intranasally. Serum was taken 7 weeks post-prime to assess F and G-specific antibodies. Statistical analysis was performed using a one-way Kruskal Wallis one-way ANOVA, followed by Dunn's multiple comparison test. p Values < 0.05 (alpha) were considered statistically significant with *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant (p > 0.05).

IFN-γ, IL-2, and TNF-α secreting cells stimulated by NiV-F and G glycoproteins
Splenocytes (Fig. 8A) and lung cells (Fig. 8B)    www.nature.com/scientificreports/ Five weeks post-boost, mouse splenocytes, and lungs were collected from BALB/c and AJ mice and analyzed for effector (T EM ), central (T CM ), and resident tissue (T RM ) CD4 + (Supplementary Fig. 6) and CD8⍺ + T cells (Fig. 9). Splenocytes from MVA-vaccinated BALB/c mice (Fig. 9A) had significant effector memory expression in MVA-FG IN groups compared to MVA-FG SC (p < 0.0001), and naive mice (p < 0.0001). Mice vaccinated with MVA-GFP SC also showed significant expression of effector memory compared to naive mice (p = 0.0053). For central and resident memory CD8⍺ + T cells, no significant differences were found, however, MVA-FG IN mice had the highest level of expression compared to MVA-FG SC, MVA-GFP SC, and naive mice. Lung cells (Fig. 9B) were collected from BALB/c mice vaccinated with MVA-FG SC and IN and MVA-GFP SC with low cell collection in naive mice being excluded from the data set. MVA-FG IN mice had the highest expression of all memory CD8⍺ + T cell subsets in the MVA-FG IN group, significantly greater than the MVA control SC group for effector memory expression (p = 0.0455). MVA-FG SC groups also saw some effector, central, and tissue resident CD8⍺ + T cell expression, with little to no expression seen in MVA-GFP SC groups.
Splenocytes collected from AJ mice (Fig. 9C) had significantly high expression of T EM in RCN-FG SC mice compared to RCN-WT SC (p = 0.0264) and naive AJ mice (p = 0.0002). Mice vaccinated with RCN-FG IN (p = 0.0205) and RCN-WT SC (p = 0.0470) also showed significant effector memory CD8⍺ + expression compared with naive mice. While no significant differences were seen for central or tissue resident memory CD8⍺ + splenocytes, RCN-FG SC and IN mice had the higher expression of both phenotypes compared to RCN-WT SC vaccinated mice and naive AJ mice. For lung cells (Fig. 9D) from AJ mice, no significant differences were seen between groups for CD8⍺ + T cell memory subsets, however higher levels of expression was seen in mice vaccinated with RCN-FG SC and IN.
Importantly, both vaccines generated CD8ɑ + T EM , T CM, and T RM responses in lung and spleen cells from vaccinated mice upon stimulation from peptides from the F and G proteins. While the MVA-FG vaccine generated the highest level of circulating antibodies when given SC, the highest phenotypic expression of effector, central, and tissue resident CD8ɑ + T cells in both splenocytes and lung cells was detected when given intranasally which should be explored in future studies. Excitingly, the RCN-FG vaccine candidate generated effector and tissue Mucosal antibodies assessed via ELISA. BALF was collected from BALB/c and AJ mice 5 weeks postboost, and the F and G antibody titers were assessed for IgG, IgA, and IgM antibody types to see if antibodies were generated in the respiratory tract along the natural route of infection. F-specific IgG antibodies (Fig. 10A) were detected in the RCN-FG IN group at a GMT of 125 which was significantly higher than the RCN-FG SC (p = 0.0032) and the RCN-WT SC (p = 0.0032) groups which had no detectable antibodies. For F-specific IgA (Fig. 10B), the RCN-FG IN group was the only group that had any detectable IgA antibody titers with a geometric mean of 50. For F-specific IgM (Fig. 10C), no antibodies against the F protein were detected in vaccine or vector control groups. IgG antibody titers against the G protein (Fig. 10D) (Fig. 10E) or IgM (Fig. 10F) G-specific antibodies were detected for any vaccine or vector control groups in AJ mice. As hypothesized, neither vaccine produced mucosal antibodies when vaccinated subcutaneously with one or two doses, however, while neither vaccine produced F or G-specific IgM antibodies, both vaccines had detected IgG antibody titers for intranasally vaccinated mice. Notably, high G-specific antibody titers were seen for both vaccines and the generation of F-specific IgG and IgA antibodies were detected for mice vaccinated with RCN-FG. Interestingly, while F-specific circulating antibodies were produced, no F-specific antibodies were detected in respiratory tract of MVA-FG IN groups.  15,42 ; however, challenge studies will need to be conducted to assess protection and efficacy.

Discussion
Regarding the cellular immune response, splenocytes and lung cells harvested from mice vaccinated with MVA-FG and RCN-FG vaccine candidates secreted TNF-⍺, IFN-γ, and IL-2 in response to F and G protein stimulation. MVA-vaccinated mice had higher levels of cytokine expression compared to RCN-vaccinated mice. MVA-FG mice vaccinated IN had the highest expression of TNF-⍺, IFN-γ, and IL-2 in splenocytes. Furthermore, CD4 + and CD8ɑ + T cell memory responses showed the generation of T EM , T CM, and T RM in both MVA-FG and RCN-FG vaccinated mice. Specifically, the MVA-FG vaccine given intranasally saw the highest phenotypic expression of effector, central, and tissue resident CD8ɑ + T cells in both splenocytes and lung cells compared to vector control and naive mice. The RCN-FG vaccine candidate generated high effector memory CD8ɑ + T cells when given SC and produced tissue resident CD8ɑ + T cells via IN administration in splenocytes. In lung cells, the RCN-FG vaccine showed higher effector, central, and tissue resident CD8ɑ + T cell expression compared with RCN-WT SC and naive mice. Again, while challenge studies would need to be conducted to assess protection, cytokine secretion and the generation of T EM , T CM , and T RM in both spleen and lung cells are comparable to levels seen in swine challenge studies that confer protection 43 .
Recombinant poxvirus vectors are easily constructed and provide high immunogenicity and enhance the induction of cellular and humoral-mediated immune responses 44 . Furthermore, unlike some viral vectors, poxviruses do not carry the risk of DNA recombination in target cell genomes due to the characteristic of cytoplasmic replication. While the consideration of vector immunity is an important area of focus, studies have shown that a single recombinant poxvirus can successfully express transgenes after multiple immunizations 45 . It is hypothesized that the antibodies produced against poxvirus target the virion during release from infected cells and not the entry/attachment phase at initial infection 45 . With this is mind, people recently infected with poxvirus would generate antibodies that prevent cell-to-cell spread of the viral vectors at the entry/attachment phase 45 . With one of the last known cases of smallpox in Bangladesh and the deployment of the MVA vaccine to counteract recent monkeypox infections in humans, long-term vector immunity should be studied in greater depth 46 . In addition, the poxvirus viral vectors used as the delivery platform for the NiV F and G proteins are safe and effective [18][19][20][21][23][24][25][26][27][28][29] . In wildlife, both the MVA and RCN viral vectors are safe and effective in bat and pig populations 47,48 . In this study, mice vaccinated with the MVA-FG and RCN-FG vaccines saw limited weight loss post-vaccination suggesting an initial safe vaccine profile. However, further preliminary data would need to be collected to determine the final safety profile prior to clinical trials or animal challenge studies such as cytokine production, and monitoring for immediate or long-term adverse events.
Additionally, the MVA and RCN viral vectors elicit high humoral immune responses in bats through oral immunization necessary for practical, wide-scale animal vaccinations in the field 49 . The RCN vectored vaccine presents a promising wildlife vaccine against NiV in both the bat reservoir and pigs, an amplifying host of NiV, to reduce the spread from wildlife to human populations and prevent the economic disruption caused by swine exportation to areas without the competent host reservoir. By being able to vaccinate both humans and wildlife, www.nature.com/scientificreports/ www.nature.com/scientificreports/ the MVA vaccine candidate presents a possible One-Health vaccine candidate and creates an avenue for disease eradication. Current vaccine approaches for Nipah rely on subunit, mRNA, VLP, DNA, or viral vector strategies [48][49][50][51][52][53][54][55][56][57] . Currently, an Equivac ® HeV recombinant subunit vaccine, licensed for horses in Australia against Hendra virus and NiV, is undergoing phase I clinical trials for humans 48,58 . Like the vaccines proposed in this study, the Equivac® HeV vaccine focuses on a One-Health approach against Hendra virus by targeting the transmission from horses to humans 59 . While this vaccine has proven to protect both ferrets and African green monkeys from fatal NiV challenges, only partial protection was obtained in pigs due to their requirement of both cellular and humoral immune responses for complete protection 60 . Recombinant vectored vaccines have many promising candidates in development tailored toward both humans and pigs including vaccines such as the recombinant rabies virus (NIPARAB), a vesicular stomatitis virus (rVSV-ΔG-NiV B G), canarypox virus (ALVAC-F/G) vaccines, and ChAdOx1 NiV B , a simian adenovirus vectored vaccine 53,[61][62][63] . Although promising, vaccine candidates against NiV fail to include the reservoir host as a vaccine target, but instead focus on the transmission between humans and swine. Due to the infrequent nature of NiV outbreaks and limited wide-scale marketability, vaccination campaigns may focus on rapid vaccine deployment in outbreak regions 64 . By deploying a vaccine capable of use in the reservoir host, wildlife vaccination can focus on preventing the spread of NiV between migratory bat regions and to other susceptible species such as pigs and humans during seasonal outbreaks. While vaccine models have been able to induce protective immune responses, few vaccine candidates (RABV and VSV) have tested protective efficacy via intranasal administration, significantly reducing their practical application in animal populations that require mucosal delivery. For current vaccine models to fully target the transmission cycle of NiV, they would need to entail a collaborative effort to halt transmission between the reservoir host, the amplifying host, and humans. Without a One Health-based vaccine approach capable of targeting the transmission cycle of NiV, control and prevention focus on mitigation after an outbreak occurs. Since NiV persists in bat reservoirs, this reservoir must be targeted through vaccine strategies to prevent wide-scale outbreaks.
These results present two promising poxvirus vaccine candidates against NiV that may provide an avenue for protection in both humans and animals involved in NiV transmission. The stimulation of high humoral immune responses through both subcutaneous and intranasal routes highlights their potential as wildlife vaccines in humans, swine, and the reservoir bat species. Finally, the induction of CD8 + T cell responses emphasizes the induction of cellular immune responses required for complete protection against NiV in swine species.